The long-term objective of this of this project is to understand the biological function, mechanism and structure of the eukaryotic oligosaccharyltransferase (OST). The OST transfers a preassembled high mannose oligosaccharide onto asparagine residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period particular emphasis will be placed on elucidating the in vivo roles of two isoforms of the human oligosaccharyltransferase that are composed of an active site subunit (STT3A or STT3B) and a shared set of non-catalytic subunits. The two OST isoforms differ greatly both with respect to specific activity and stringency of dolichol-linked oligosaccharide donor selection. STT3A and STT3B are widely expressed in human tissues, and are coexpressed in all cell lines analyzed to date. We will test the hypothesis that mammalian OST isoforms have overlapping but non-identical roles in N-linked glycosylation of proteins. Hypoglycosylation of glycoprotein reporters will be analyzed in cells that have been manipulated to express a single OST isoform. Simultaneous siRNA mediated knockdowns of an OST active site subunit (STT3A or STT3B) and the ALG6 glucosyltransferase will provide a model system to analyze the influence of OST isoform expression on protein hypoglycosylation in cells that are impaired in dolichol-linked oligosaccharide assembly. Protein sequence database searches predict that most protist organisms assemble monomeric or hetero-tetrameric OST complexes instead of the heptameric or octameric OST complexes assembled by fungi and metazoan organisms. Novel assays and biochemical probes will be used to analyze the binding of dolichol-linked oligosaccharide donor and peptide acceptors to the OST from diverse eukaryotic organisms including the pathogenic protists Trypanosoma cruzi, Entamoeba histolytica and Trichomonas vaginalis and the pathogenic yeast Cryptococcus neoformans. A final goal of this project is to obtain a mid to high-resolution structure of the mammalian OST by electron crystallography. The research proposed here will provide insight into the family of inherited human autosomal diseases known as congenital disorders of glycosylation (CDG-I). Analysis of protein N-glycosylation in pathogenic protists may reveal differences in enzymatic reaction mechanism that could potentially be exploited to produce useful pharmacological agents.